Droplet interfaces in electro-wetting devices

ABSTRACT

Droplet interfaces are formed between droplets in an electro-wetting device comprising an array of actuation electrodes. Actuation signals are applied to selected actuation electrodes to place the droplets into an energised state in which the shape of the droplets is modified compared to a shape of the droplets in a lower energy state and to bring the two droplets into proximity. The actuation signals are then changed to lower the energy of the droplets into the lower energy state so that the droplets relax into the gap and the two droplets contact each other thereby forming a droplet interface. The use of sensing electrodes in the device permit electrical current measurements across the droplet interface. The sensing electrodes can be used for either (i) applying a reference signal during droplet actuation or (ii) recording electrical current measurements.

In some aspects, the present invention relates to electro-wettingdevices and their use to form droplet interfaces between droplets ofliquid in a fluid medium. In some aspects, the present invention relatesto electro-wetting devices and their use is making measurements ondroplet interfaces formed using electro-wetting.

Electro-wetting devices, for example electro-wetting on dielectric(EWOD) devices, are known for manipulating droplets of liquid in a fluidmedium.

Considering this in more detail, electro-wetting on dielectric is awell-known technique for manipulating droplets of fluid by theapplication of an electric field, for example as disclosed inUS2016/0305906. Example configurations and operation of EWOD devices aredescribed in the following documents. U.S. Pat. No. 6,911,132 disclosesa two-dimensional EWOD array to control the position and movement ofdroplets in two dimensions. U.S. Pat. No. 6,565,727 discloses methodsfor other droplet operations including the splitting and merging ofdroplets, and the mixing together of droplets of different materials.U.S. Pat. No. 7,163,612 describes how an active matrix (AM) arrangementbased on thin film electronics including thin-film transistors (TFT) maybe used to control the addressing of voltage pulses to an EWOD device,using circuit arrangements similar to those employed in AM displaytechnologies. Devices of this general type may be referred to as AM-EWODdevices.

It has been proposed to use EWOD devices to manipulate such droplets toform droplet interfaces between droplets, for example comprising amembrane of amphipathic molecules. That provides a potentially usefulsystem for studying the droplet interfaces themselves, and alsoprocesses occurring at the droplet interfaces. In one example havingparticular interest, such processes may involve insertion of atransmembrane pore, and subsequent measurement of properties such as ioncurrent flow that may be dependent on interaction of an analyte withsuch a transmembrane pore.

Membranes of amphipathic molecules, for example artificial planar lipidbilayers may serve as simplified models of biological membranes and arewidely used for the study of various processes, including thecharacterisation of transmembrane pores, such as transmembrane proteinpores, for example ion-channels. Ion-channels are a diverse group oftransmembrane protein pores that in biology selectively control themovement of specific ions across cell membranes, establishing voltageand electrochemical gradients that are fundamental to a wide variety ofbiological processes.

Single-channel recording (SCR) of individual protein pores is a powerfulmeans of studying channel protein function. Single-channel recordingmeasures changes in ion-current through single protein channels, and canexamine voltage dependence, gating behaviour, ligand binding affinity,and ion selectivity at the single-molecule level. Various methods may beemployed to form lipid bilayers such as disclosed by Montal, M. &Mueller, P. 1972. Proceedings of the National Academy of Sciences of theUnited States of America 69, 3561-3566). Although widely used, planarlipid bilayers are difficult to prepare, and their short lifetimeprohibits their use in many situations.

Thus other membranes of amphipathic molecules have been proposed.Alternatives to planar lipid bilayers are disclosed, for example,WO-2008/012552 which discloses a method of forming bilayers ofamphipathic molecules uses droplets of aqueous solution in a hydrophobicmedium such as oil.

Arrays of individual suspended membranes of amphipathic moleculescontaining respective ion channel protein pores or nanopores may beprovided, for example disclosed in WO-2014/064443. Ion current betweentwo aqueous solutions provided at either side of the amphipathicmembrane may be measured in order to characterise an analyte such aspolynucleotide and commercial devices such as the MinION™ comprising ananopore array that is able to determine a polynucleotide sequence aresold by Oxford Nanopore Technologies Ltd.

Droplet interfaces between droplets in contact with one another aretherefore an alternative way of forming membranes of amphipathicmolecules. Such a membrane formed by a bilayer of amphipathic moleculesmay be referred to as a droplet interface bilayer (DIB). Multiplemembranes can be formed at the interface between multiple pairs ofdroplets. Techniques for forming DIBs are disclosed for example inLeptihn et al, Nature Protocols 8, 1048-1057 (2013) and DIBs may be usedfor the study of transmembrane pores inserted therein. For example,Martel et al., Biomicrofluidics 6, 012813 (2012) discloses amicrofluidic device for forming droplet interface bilayers into which aprotein channel was inserted. Gold microwires were deposited onto thesubstrate including the actuation electrodes upon which Ag/AgCl padswere provided in order to make electrical contact with each droplet inorder to carry out measurements of ion current flow through the membranechannel.

The first aspect of the present invention is concerned with methods anddevices for forming a droplet interface in an electro-wetting device.

According to a first aspect of the present invention, there is provideda method of forming a droplet interface in an electro-wetting device,the electro-wetting device comprising: an array of actuation electrodes;an insulator layer covering the actuation electrodes and having anoutermost hydrophobic surface; disposed on the hydrophobic surface, afluid medium and two droplets comprising liquid in the fluid medium, oneof the liquid and the fluid medium being polar, and the other of theliquid and the fluid medium being apolar, whereby the actuationelectrodes are capable of electro-wetting the droplets when actuationsignals are applied thereto, the method comprising: applying actuationsignals to selected actuation electrodes to place one or both of the twodroplets in an energised state in which the shape of said one or bothdroplets is modified compared to when in a lower energy state and tobring the two droplets into proximity with a gap therebetween, the gapbeing chosen such that the two droplets do not contact each other whenone or both are in the energised state and contact each other to form adroplet interface when in the lower energy state; and changing theactuation signals applied to the actuation electrodes to lower theenergy of said one or both droplets into the lower energy state so thatsaid one or both droplets relax into the gap and the two dropletscontact each other thereby forming a droplet interface.

This method is applied to an electro-wetting device that comprises anarray of actuation electrodes and an insulator layer covering theactuation electrodes and having an outermost hydrophobic surface. Insuch an electro-wetting device, a fluid medium and two dropletscomprising liquid in the fluid medium may be disposed on the hydrophobicsurface. The actuation electrodes are capable of electro-wetting thedroplets when actuation signals are applied thereto, thereby allowingmanipulation of the droplets by selection of the actuation signals.

The method provides a reliable technique for forming a droplet interfacebetween two droplets. With the present method the actuation signals areapplied to the actuation electrodes in two phases. In the initial phase,the applied actuation signals have a pattern selected to place one orboth of the two droplets in an energised states. As a result, the shapeof one or both droplets is modified compared to when in a lower energystate. In such an energised state, the two droplets are brought intoproximity with a gap therebetween. The gap is chosen such that the twodroplets do not contact each other when one or both are in the energisedstate and contact each other to form a droplet interface when in thelower energy state.

In a subsequent phase, the applied actuation signals are changed tolower the energy of said one or both droplets into a lower energy state.As a result, one or both droplets relax into the gap and the twodroplets contact each other. A droplet interface is thereby formedbetween the two droplets. Thus, movement of a surface of the one or bothdroplets is caused by relaxation of the energised state, which is apassive process.

This process improves the reliability of formation of the dropletinterface, compared to attempting to bring two droplets into contactdirectly by applying actuation signals that move the entire dropletstowards one another. While such methods might be possible in principle,the droplets have a tendency to fuse (i.e. merge) and it is difficult tomaintain the interface between the droplets.

While the method may be applied by placing a single one of the twodroplets in an energised state, preferably both droplets are placed inthe energised state. As a result, the surfaces of both droplets relaxinto the gap and contact each other to form the droplet interface. Inthis manner, relaxation of both droplets is used to form the dropletinterface, which further increases the reliability of formation.

In the energised state of the one or both droplets, any shape of thedroplets may be selected that allows the desired relaxation of thesurface to form the droplet interface. While various shapes arepossible, advantageously the shape of the droplet in the energised stateas viewed in the plane of the electro-wetting device is elongate.Similarly, the shape of the contact line of the droplet in the energisedstate is elongate. In that case, the gap between droplets may extendalong a major length of the elongate shape, so that on relaxation, asurface of the droplet extending along the major length may move intothe gap to contact the other droplet.

Where an elongate shape is used, the shape of the droplet in theenergised state may have an aspect ratio of at least 2:1, preferably atleast 4:1 or at least 8:1. In general, increasing the aspect ratioincreases the degree of movement of the surface of the at least onedroplet, which assists bringing the droplets into contact.

During the step of applying actuation signals to the actuationelectrodes, the two droplets may be brought into proximity with thecentroids of the two droplets, separated by a distance less than thecombined radii of the droplets, along a line between the two centroidsin the lower energy state of the droplets.

The method may be applied with advantage to an electro-wetting devicewherein the area enclosed by the contact line of the droplets in thelower energy state covers at least two actuation electrodes, preferablyat least 5 actuation electrodes, at least 10 actuation electrodes or atleast 20 actuation electrodes. In general, in the design of theelectro-wetting device, the more actuation electrodes a droplet coversthe better the resolution of the control of the shape in the energisedstate of the droplet. That in turn allows the degree of movement of thesurface of the at least one droplet to be increased, which assistsbringing the droplets into contact.

The actuation signals that are selected to energise the one or moredroplets may be alternating (AC) actuation voltage signals. In general,the use of AC actuation signals in an electro-wetting device is known tobe advantageous for manipulating droplets. In that case, preferably, thestep of changing the actuation signals applied to the selected actuationelectrodes may comprise applying DC potentials or floating potentials tothe selected actuation electrodes in place of the AC actuation signals.This improves the reliability of formation of the droplet interfaces,because the DC potentials or floating potentials are less likely torupture the droplet interface than if AC actuation signals weremaintained.

The method may be applied to an electro-wetting device wherein theinsulator layer comprises a layer of electrically insulating materialcoated by a hydrophobic material that forms said hydrophobic surface.

The method may be applied to an electro-wetting device that furthercomprises a second substrate facing the hydrophobic surface of theinsulator layer, wherein the second substrate is coated by a hydrophobicmaterial forming a further hydrophobic surface facing the hydrophobicsurface of the insulator layer. In this case, the droplets may bedisposed on the further hydrophobic surface of the hydrophobic layer aswell as the hydrophobic surface of the insulator layer. In this manner,the droplets are sandwiched between the two substrates, which constrainsthe shape of the droplets. This improves the degree of control of theshape of the droplets between the energised state and lower energystate, which in turn improves the reliability of the formation ofdroplet interfaces.

Furthermore, the second substrate may support sensor electrodes thatmake an electrical connection with the droplets between which a dropletinterface is formed.

The method may be applied to an electro-wetting device that furthercomprises an active matrix arrangement connected to the actuationelectrodes.

The method may be applied to form only a single droplet interfacebetween two droplets, but equally the method may be applied with one ormore further droplets disposed on the hydrophobic surface, and the stepsof applying and changing actuation signals to the actuation electrodesmay be performed to form plural droplet interfaces between plural pairsof droplets.

After formation of a droplet interface, electrical measurements may betaken between the droplets across the droplet interface. For example,the electrical measurements may be measurements of ion flow betweendroplets through a transmembrane pore and/or may be taken while applyinga potential difference between the droplets.

Further according to the first aspect of the present invention, there isprovided an electro-wetting device for forming a droplet interface whichimplements a similar method to that described above.

The second aspect of the present invention is concerned with makingelectrical connections to respective droplets in a system of dropletsformed in an electro-wetting device and having one or more dropletinterfaces between droplets. Such electrical connections may have thepurpose of sensing a property of the droplets, such as the size orlocation of a droplet when performing various droplet operations in theelectro-wetting device, or the purpose of taking measurements across adroplet interface.

US-2010/0,194,408 discloses a method, circuit and apparatus fordetecting capacitance on a droplet actuator inter alia for determiningthe presence, partial presence or absence of a droplet at an actuationelectrode. U.S. Pat. No. 8,653,832 describes how an impedance (orcapacitance) sensing function can be incorporated into the array elementcircuit of each array element of an AM-EWOD device, wherein theimpedance sensor circuit may be used for determining the presence andsize of droplets present at each electrode in the array. However, theseapproaches are limited by the need to obtain signals from the sameelectrodes to which the actuation signals are applied.

Martel et al., Biomicrofluidics 6, 012813 (2012) discloses amicrofluidic device for forming droplet interface bilayers into which aprotein channel was inserted, wherein gold microwires were depositedonto the substrate including the actuation electrodes upon which Ag/AgClpads were provided in order to make electrical contact with each dropletin order to carry out measurements of ion current flowing through themembrane channel. However, this construction is inconvenient anddifficult to manufacture, as well as limiting the reliability of takingmeasurements and limiting the scalability of the technique.

According to a second aspect of the present invention, there is providedan electro-wetting device for taking measurements across a dropletinterface, the electro-wetting device comprising: a first substratesupporting an array of actuation electrodes; an insulator layer coveringthe actuation electrodes and having a hydrophobic surface, a secondsubstrate facing the hydrophobic surface of the insulator layer andsupporting at least one set of at least two sensor electrodes, theelectro-wetting device being arranged to receive a fluid medium anddroplets comprising liquid in the fluid medium disposed on thehydrophobic surface, the actuation electrodes being configured toreceive actuation signals for electro-wetting received droplets in orderto form at least one system of droplets having one or more dropletinterfaces between droplets, and the sensor electrodes of each set beingconfigured to make electrical connections to respective droplets in theat least one system of droplets.

Thus, in the electro-wetting device, sensor electrodes are provided on asecond substrate facing the facing the hydrophobic surface of theinsulator layer that covers the actuation electrodes. Such sensorelectrodes are arranged in at least one set of at least two sensorelectrodes, and the sensor electrodes of each set are configured to makeelectrical connections to respective droplets in a system of droplets.This provides a convenient and reliable way to make electricalconnections to the droplets.

The second substrate may be coated by a hydrophobic material forming afurther hydrophobic surface facing the hydrophobic surface of theinsulator layer. In that case, the electro-wetting device may bearranged to receive the fluid medium and the droplets disposed on thefurther hydrophobic surface of the hydrophobic layer as well as thehydrophobic surface of the insulator layer. In this manner, the dropletsare sandwiched between the two substrates, which constrains the shape ofthe droplets. This improves the degree of control of the droplets byactuation signals applied to the actuation electrodes.

Where second substrate is coated by a hydrophobic material, then thehydrophobic material coating the second substrate may have aperturesexposing at least part of the sensor electrodes. This improves theelectrical connection between the sensor electrodes and the droplets.

The second substrate further supports at least one further electrode,for example for receiving a reference signal while actuation signals areapplied to the actuation electrodes for manipulating the droplets.

The sensor electrodes and the further electrodes, where provided, may bedeposited on a surface of the second substrate facing the firstsubstrate. In that case, the further electrodes, where provided, mayextend around the sensor electrodes.

The electro-wetting device may further comprise a control system that isconnected to the actuation electrodes and is configured to applyactuation signals to the actuation electrodes for manipulating receiveddroplets.

The control system may be configured, while applying actuation signalsto the actuation electrodes, to apply a reference signal to the sensorelectrodes and/or to the further electrodes, where provided.

The control system may be configured to apply actuation signals to theactuation electrodes selected to form a droplet interface at theinterface between two droplets.

The electro-wetting device may further comprise a sensor systemconnected to the sensor electrodes and configured to take electricalmeasurements, for example including impedance measurements, betweensensor electrodes that are electrically connected to respective dropletsforming a droplet interface therebetween. Such electrical measurementsmay be taken across a droplet interface between two droplets in a systemof droplets.

The sensor system may be configured to take electrical measurementsbetween respective sensor electrodes that make contact betweenrespective droplets across a droplet interface comprising a membrane ofamphipathic molecules having a transmembrane pore inserted therein, forexample measurements of ion flow between droplets through atransmembrane pore and/or electrical measurements that are dependent onan analyte that interacts with the transmembrane pore.

The sensor system may further comprise an analysis system configured toprocess the electrical measurements to analyse an analyte that interactswith the transmembrane pore. For example, where the analyte is a polymercomprising polymer units, the analysis system may be configured toprocess the electrical measurements to derive estimated identities ofthe polymer units of the polymer.

The third aspect of the present invention is concerned with use of anelectro-wetting device to perform experiments on droplet interfaces.

According to a third aspect of the present invention, there is providedan apparatus for performing experiments on droplet interfaces, theapparatus comprising: an electro-wetting device comprising an array ofactuation electrodes and an insulator layer covering the actuationelectrodes and having an outermost hydrophobic surface, theelectro-wetting device being arranged to receive a fluid medium anddroplets comprising liquid in the fluid medium disposed on thehydrophobic surface, a control system configured to apply actuationsignals to the actuation electrodes selected to manipulate receiveddroplets and to form at least one system of droplets having one or moredroplet interfaces between the droplets; and a sensor system configuredto take electrical measurements between droplets in a formed systemacross droplet interfaces.

Such an apparatus is suitable for performing experiments on dropletinterfaces.

The apparatus includes an electro-wetting device in which dropletinterfaces may be formed. The electro-wetting device comprises an arrayof actuation electrodes and an insulator layer covering the actuationelectrodes and having an outermost hydrophobic surface. Theelectro-wetting device can receive a fluid medium and dropletscomprising liquid in the fluid medium disposed on the hydrophobicsurface

The apparatus further includes a control system configured to applyactuation signals to the actuation electrodes selected to manipulatereceived droplets and to form at least one system of droplets having oneor more droplet interfaces between the droplets. Therefore, use of thecontrol system allows droplet interfaces to be formed in theelectro-wetting device.

The apparatus further includes a sensor system configured to takeelectrical measurements between droplets in a formed system acrossdroplet interfaces, thereby allowing experiments to be performed on theformed droplet interfaces.

After formation of a droplet interface, various types of electricalmeasurements may be taken between the droplets across the dropletinterface. For example, the electrical measurements may be measurementsof ion flow between droplets through a transmembrane pore and/or may betaken while applying a potential difference between the droplets.

The sensor system may further comprise an analysis system configured toprocess the electrical measurements to analyse an analyte that interactswith the transmembrane pore. For example, where the analyte is a polymercomprising polymer units, the analysis system may be configured toprocess the electrical measurements to derive estimated identities ofthe polymer units of the polymer.

Advantageously, the control system may be arranged to modify the atleast one formed system of droplets in response to the electricalmeasurements taken by the sensor system. The ability of the apparatus tomodify the formed system of droplets based on feedback from the sensorsystem provides significant advantages, because it allows the apparatusto perform experiments on droplet interfaces in an adaptive manner.

The outputs of the sensor system to which the control system respondsmay include the electrical measurements themselves. This provides afirst type of control of the experiments being performed based on theelectrical properties being measured. As the electrical properties arefundamental to the relevant processes such as formation of dropletinterfaces and reactions occurring there, this first type of controlallows those processes to be considered and adaptively modified.

Alternatively, where the sensor system comprises an analysis systemconfigured to process the electrical measurements, and said outputs ofthe sensor system include outputs of the analysis system. This providesa second type of control of the experiments being performed based on theanalysis. As such analysis allows higher level information to beobtained, for example concerning analyte being analysed, this secondtype of control provides powerful experimental adaption based on theresults of the analysis.

The types of control which may be performed are extensive, therebyproviding a powerful experimental apparatus. Some non-limitativeexamples are as follows.

The formed system of droplets may be modified by separating a dropletinterface in the system.

The formed system of droplets may be modified by moving a new dropletinto contact with a current droplet in the system of droplets andforming a droplet interface between the new droplet and the currentdroplet.

The formed system of droplets may be modified by moving a new dropletinto contact with a current droplet in the system of droplets and fusingthe new droplet and the current droplet. In that case, it may be thatthe new droplet does not comprise amphipathic molecules at the interfacebetween the liquid of the droplet and the fluid medium.

Advantageously, the control system may be arranged to apply actuationsignals to the actuation electrodes selected to form plural systems ofdroplets in parallel. This allows the apparatus to perform experimentson the plural systems in parallel with each other, thereby increasingthe experimental throughput of the apparatus.

The apparatus may further include a droplet preparation systemconfigured to form droplets disposed on the hydrophobic surface of theelectro-wetting device in the fluid medium. In this case, the controlsystem may be configured to control the droplet preparation system toform the droplets. This increases the experimental power of theapparatus as it allows droplets containing appropriate reagents to beformed for experimental purposes.

The first to third aspects of the present invention may be implementedtogether, for example in the same device or apparatus. Accordingly, thepreferred features of the first to third aspects of the presentinvention, some of which are defined in the dependent claims below, maybe combined in any combination.

To allow better understanding, embodiments of the present invention willnow be described by way of non-limitative example with reference to theaccompanying drawings, in which:

FIG. 1 is a schematic view of an apparatus including an AM-EWOD device;

FIG. 2 is a schematic perspective view of the AM-EWOD device;

FIG. 3 is a cross-sectional side view of a portion of the AM-EWODdevice;

FIGS. 4A and 4B are diagrams of a circuit representation of theelectrical load presented at the actuation electrode when a liquiddroplet is present and not present, respectively;

FIG. 5 is a plan view of thin film electronics in the AM-EWOD device;

FIG. 6 is a diagram of an array element circuit of the AM-EWOD device;

FIG. 7 is a plan view of a layer of conductive material formed on asecond substrate of the AM-EWOD device;

FIGS. 8 to 12 are plan views of a pair of droplets on an array ofactuation electrodes in the AM-EWOD device during successive stages of amethod of forming a droplet interface between the droplets;

FIGS. 13 and 14 are plan views of respective ways of forming two systemsof three droplets;

FIGS. 15 and 16 are plan views of respective systems of droplets.

FIG. 17 is two images of an array of actuation electrodes in the AM-EWODdevice;

FIG. 18 is a diagram of a patterned layer of conductive material in theAM-EWOD device;

FIG. 19 is an image of an array of actuation electrodes in the AM-EWODdevice on which three systems of two droplets are formed;

FIG. 20 is images of two sets of three droplets before and afterformation of two droplet interfaces therebetween;

FIG. 21 is a plot of electrical measurements of current and voltageduring formation, separation and re-formation of droplet interfaces inthe AM-EWOD device;

FIG. 22 is an image of a system of two droplets in the AM-EWOD device;

FIG. 23 is a trace of a current signal obtained in the system ofdroplets in FIG. 22;

FIG. 24 is a plot of electrical measurements of current and voltageduring an experiment in which DNA translocates through a pore in asystem of two droplets in the AM-EWOD device; and

FIG. 25 is a plot of electrical measurements of current for sixtranslocation events.

OVERALL APPARATUS

FIG. 1 illustrates an apparatus 30 for forming droplet interfaces andfor performing experiments thereon. The apparatus 30 includes a reader32 and a cartridge 33 that may be inserted into the reader 32. Thecartridge 33 contains an AM-EWOD device 34 which is an example of anelectro-wetting device. The AM-EWOD device 34 is shown in FIG. 2 anddescribed further below.

The reader 32 and cartridge 33 may be electrically connected togetherwhile in use, for example by a cable of connecting wires 42, althoughvarious other methods (e.g. wireless connection) of providing electricalcommunication may be used.

The reader 32 also comprises a droplet preparation system 35 configuredto form droplets 1 comprising liquid in a fluid medium 60 in the AM-EWODdevice 34 when the cartridge 33 is inserted. Suitable materialproperties for the droplets 1 and the fluid medium 60 are discussedbelow. The droplet preparation system 35 may also be able to carrysample preparations to prepare an analyte to be measured, alternativelysample preparation may be carried out in the AM-EWOD device 34. Thesamples may be compartmentalised for a library preparation or forsequencing.

The droplet preparation system 35 may comprise fluid input ports thatperform the function of inputting liquid into the AM-EWOD device 34 fromone or more reservoirs and thereby generating droplets within AM-EWODdevice 34. The droplet preparation system 35 may be formed byconventional fluidics elements, for example controlling flow of liquidby electro-wetting. The droplet preparation system 35 desirably has theability to accurately control the volumes of created droplets 1,typically accurate to 2-3%. Typically, the droplets may have respectivevolumes between 1 nL and 10 μL

The apparatus 30 further includes a control system 37 provided in thereader 32. In this example, the control system 37 includes controlelectronics 38 and a storage device 40 that may store any applicationsoftware any data associated with the system. The control electronics 38may include suitable circuitry and/or processing devices that areconfigured to carry out various control operations relating to controlof the AM-EWOD device 34, such as a CPU, microcontroller ormicroprocessor.

Among their functions, to implement the features of the presentinvention, the control electronics 38 may comprise a part of the overallcontrol system 37 that may execute program code embodied as a controlapplication within the storage device 40. The storage device 40 may beconfigured as a non-transitory computer readable medium, such as randomaccess memory (RAM), a read-only memory (ROM), an erasable programmableread-only memory (EPROM or Flash memory), or any other suitable medium.Also, while the code may be executed by control electronics 38 inaccordance with an exemplary embodiment, such control systemfunctionality could also be carried out via dedicated hardware,firmware, software, or combinations thereof.

As described in more detail below, the control system 37 is configuredto perform control of various elements of the apparatus 1, includingcontrol of the droplet preparation system 35, to form the droplets 1 andcontrol of the application of actuation signals for manipulatingdroplets. In particular, the control system 37 is configured to form oneor more systems of two or more droplets 1. Within the or each system ofdroplets 1, one or more droplet interfaces are formed between respectivepairs of droplets 1. The control system 37 may also provide a graphicaluser interface (GUI) to a user which provides for the user to input ofprogram commands such as droplet operations (e.g. move a droplet), assayoperations (e.g. perform an assay), and for displaying the results ofsuch operations to the user.

Electro-Wetting Device

FIGS. 2 and 3 illustrate the AM-EWOD device 34.

As seen in FIG. 2, the AM-EWOD device 34 has a first substrate 44 (whichis the lowermost substrate in FIGS. 2 and 3) with thin film electronics46 disposed upon the first substrate 44. An array 50 of actuationelectrodes 48 are supported by the first substrate 46 on top of the thinfilm electronics 46. The thin film electronics 46 are arranged to drivethe actuation electrodes 48.

The array 50 of actuation electrodes 48 may be an X by Y rectangulararray, where X and Y are any integers. The actuation electrodes 48 maybe formed, for example, from indium tin oxide (ITO) or anothertransparent metal oxide, or a metal, or any other electricallyconductive material.

The AM-EWOD device 34 has a first substrate also includes a secondsubstrate 54 (which is the uppermost substrate in FIGS. 2 and 3)separated by a spacer 56 from the first substrate 44. As describedfurther below, droplets 1 are disposed between the first substrate 44and a second substrate 54. A single droplet is shown in FIGS. 2 and 3but in general multiple droplets 1 are present.

The layered structure of the AM-EWOD device 34 is best seen in FIG. 3which illustrates a portion thereof including two actuation electrodes48 supported by the first substrate 44. The actuation electrodes 48 maybe formed from a patterned layer of conductive material.

An insulator layer 61 comprising a layer 62 of electrically insulatingmaterial coated by a hydrophobic material 64 is disposed on the firstsubstrate 44, covering the actuation electrodes 48. The hydrophobicmaterial 64 forms an outermost hydrophobic surface of the insulatorlayer 61.

The second substrate 54 faces the hydrophobic surface of the insulatorlayer 61. The second substrate 54 supports a layer 58 of conductivematerial that is deposited on the surface of the second substrate 54facing the insulator layer 61. The layer 58 of conductive material ispatterned to form more electrodes, as described in more detail.

The second substrate 54 is coated by a hydrophobic material 68 thatcovers the layer 58 of conductive material and forms a furtherhydrophobic surface facing the hydrophobic surface of the insulatorlayer 61.

The hydrophobic materials 64 and 68 may be formed by any suitablematerials (which may be the same or different), for example afluoropolymer.

The droplets 1 are received in the AM-EWOD device 34, disposed within afluid medium 60. The droplets 1 and the fluid medium 60 are disposed onthe hydrophobic surface of the insulator layer 61 and on the furtherhydrophobic surface of the hydrophobic material 68 that coats the secondsubstrate 54. In this manner, the droplets 1 are sandwiched between thefirst and second substrates 44 and 54, which constrain the shape of thedroplets 1. This improves the degree of control of the droplets 1 by theactuation signals applied to the actuation electrodes 48 in the mannerdescribed below.

The droplets 1 have a contact angle 66 with the hydrophobic surface ofthe insulator layer 61. The contact angle 66 is determined by thebalancing of the surface tension components (1) from the hydrophobicsurface to the liquid of the droplets 1 (Γ_(SL)) interface, (2) from theliquid of the droplets 1 to the surrounding fluid medium 60 (Γ_(LG))interface, and (3) from the hydrophobic surface to the surrounding fluidmedium 60 (Γ_(SG)) interface. Where no voltages are applied, the contactangle 66 satisfies Young's law, and is of size 0 given by the equationcos θ=((Γ_(SG)−Γ_(SL))/Γ_(LG)).

Accordingly, the actuation electrodes 48 are capable of electro-wettingthe droplets 1 when actuation signals are applied to the actuationelectrodes 48. The actuation signals create electrical forces thateffectively control the hydrophobicity of the hydrophobic surface of theinsulating layer 61, and thereby energise the droplet 1. In such anenergised state, the droplets 1 will have a shape that is modifiedcompared to when the droplets 1 are in a lower energy state, i.e. astate in which the actuation signals provide less or no energy to thedroplets 1.

Such references to the shape being modified may refer to the shape inthe plane of the AM-EWOD device 34, i.e. parallel to the hydrophobicsurface of the insulator layer 61. Although energy supplied by theactuation signals will modify the three-dimensional shape of thedroplets 1, the shape is most greatly affected in the plane of theAM-EWOD device 34, being the direction in which the actuation electrodes48 are arrayed.

Similarly, references to the shape being modified compared to when in alower energy state may refer to the shape of the contact line of thedroplets 1. In this context, the term “contact line” has its normalmeaning of the line along which the interface between the droplet 1 andthe fluid medium 60 contacts the hydrophobic surface of the insulatinglayer 61 above the actuation electrodes 48.

By selective control of the pattern of actuation signals applied to theselected actuation electrodes 48, the droplets 1 may be manipulated andmoved in the lateral plane between the first and second substrates 44and 54. In general terms, such manipulation of droplets 1 in this mannermay apply techniques known for EWOD devices.

The actuation signals may take any form suitable for electro-wetting thedroplets 1. Typically, the actuation signals may be AC actuationsignals, but in general they could also be DC voltage potentials withrespect to a reference voltage. While applying the actuation signals, areference signal is applied to a reference electrode 59 elsewhere in theAM-EWOD device, as described further below.

The reference signal may take any suitable form. In one example, thereference signal may be a fixed reference voltage. In another examplewhere the actuation signals are AC actuation signals, the referencesignal may be an AC reference signal which is in anti-phase with the ACactuation signals. In this example, the magnitude of the potentialdifference between the actuation electrodes 48 and the referenceelectrode 59 is increased, for example being doubled when the ACactuation signals and the AC reference signal are of equal magnitude,compared to a reference signal that is a fixed reference voltage.

FIG. 4A shows a simplified circuit representation of the electrical load70A between the actuation electrode 48 and a reference electrode 59 inthe case where a droplet 1 is present. The droplet 1 can usually bemodelled as a resistor and capacitor in parallel. Typically, theresistance of the droplet 1 will be relatively low (e.g. if the dropletcontains ions) and the capacitance of the droplet will be relativelyhigh (e.g. because the relative permittivity of polar liquids isrelatively high, e.g. ˜80 if the droplet 1 is aqueous). In manysituations the droplet resistance is relatively small, such that at thefrequencies of interest for electro-wetting, the droplet 1 may functioneffectively as an electrical short circuit. The hydrophobic materials 64and 68 have electrical characteristics that may be modelled ascapacitors, and the insulating material of the layer 62 may also bemodelled as a capacitor. The overall impedance between the actuationelectrode 48 and the reference electrode 59 may be approximated by acapacitor whose value is typically dominated by the contribution of theinsulating material of the layer 62 and hydrophobic materials 64 and 68contributions, and which for typical layer thicknesses and materials maybe on the order of a pico-Farad in value.

FIG. 4B shows a circuit representation of the electrical load 70Bbetween the actuation electrode 48 and the reference electrode 59 in thecase where no droplet 1 is present. In this case the droplet componentsare replaced by a capacitor representing the capacitance of thenon-polar fluid 60 which occupies the space between the top and firstsubstrates. In this case the overall impedance between the actuationelectrode 48 and the reference electrode 59 may be approximated by acapacitor whose value is dominated by the capacitance of the non-polarfluid and which is typically small, of the order of femto-Farads.

For the purposes of driving and sensing the actuation electrodes 48, theelectrical loads 70A and 70B overall function in effect as a capacitor,whose value depends on whether a droplet 1 is present or not at a givenactuation electrode 48. In the case where a droplet is present, thecapacitance is relatively high (typically of order pico-Farads), whereasif there is no droplet 1 present the capacitance is low (typically oforder femto-Farads). If a droplet partially covers a given electrode 48then the capacitance may approximately represent the extent of coverageof the actuation electrode 48 by the droplet 1.

FIG. 5 illustrates the arrangement of the thin film electronics 46 inthe AM-EWOD device 34. The thin film electronics 46 is located on thefirst substrate 44 and comprises an active matrix arrangement of arrayelements 51 each comprising an array element circuit 72 for controllingthe electrode potential of a corresponding actuation electrode 48.Integrated row driver 74 and column driver 76 circuits are alsoimplemented in thin film electronics 46 to supply control signals to thearray element circuit 72. In this manner, the array element circuit 72may perform a function of selectively, under the control of the controlsystem 37, actuating the actuation electrode 48 to applying an actuationsignal to the actuation electrode 48. Thus, the control system 37controls the actuation signals applied to the actuation electrodes 48,such as required voltage and timing signals to perform dropletmanipulation operations.

FIG. 6 illustrates the arrangement of the array element circuit 72present in each array element 51. The array element circuit 72 containsan actuation circuit 88, having inputs ENABLE, DATA and ACTUATE, and anoutput which is connected to an actuation electrode 48.

A serial interface 82 may also be provided to process a serial inputdata stream and facilitate the programming of the required voltages tothe actuation electrodes 48 in the array 50. A voltage supply interface84 provides the corresponding supply voltages, second substrate drivevoltages, and other requisite voltage inputs. A number of connectingwires 86 between the first substrate 44 and external controlelectronics, power supplies and any other components can be maderelatively few, even for large array sizes. Optionally, the serial datainput may be partially parallelized. For example, if two data inputlines are used the first may supply data for columns 1 to X/2, and thesecond for columns (1+X/2) to M with minor modifications to the columndriver circuits 76. In this way the rate at which data can be programmedto the array elements 51 is increased, which is a standard techniqueused in Liquid Crystal Display driving circuitry.

Droplet Interface Sensing

FIG. 7 shows how the layer 58 of conductive material is patterned toform sensor electrodes 100, conductive tracks 101, and furtherelectrodes 102 which are therefore deposited on the second substrate 54and supported thereby.

The sensor electrodes 100 are arranged to make an electrical connectionwith respective droplets 1. The provision of the sensor electrodes 100is a convenient and reliable way to make electrical connections to thedroplets 1, for example to take electrical measurements between thedroplets 1 across a droplet interface formed therebetween. In contrast,such a type of electrical connection is not possible from the actuationelectrodes 58 due to the presence of the insulating layer 61 includinglayer 62 of electrically insulating material between the actuationelectrodes 48 and the droplets 1.

The hydrophobic material 68 that covers the layer 58 of conductivematerial coating the second substrate 54 is provided with apertures 69that expose part of the sensor electrodes 100, although more generallythe apertures may be larger and expose the entirety of the sensorelectrodes 100. The apertures 69 in the hydrophobic material 68 assistin making an electrical contact between the sensor electrodes 100 andthe droplets 1. The fluid medium 60 and/or the liquid of the droplets 1can flow into apertures 69, and have a lower electrical impedance thanthe hydrophobic material 68, thereby providing a conductive path.

Such apertures 69 may have the additional advantage of acting as ahydrophilic patch which helps to pin droplets 1 in position if theelectrodes are de-actuated or the device is de-powered.

Such apertures 69 may be created by selective removal of the hydrophobicmaterial 68, for example by means of a dry etch process or lift offprocess.

However, the apertures 69 are not essential and instead an electricalconnection between the sensor electrodes 100 and the droplets 1 can bemade through the hydrophobic material 68, which may be of sufficientlylow impedance (either real or imaginary parts) that an electricalmeasurement can still be taken through it. In that case, the thicknessand material properties of the hydrophobic material 68 are chosenaccordingly.

The sensor electrodes 100 are arranged in sets and the sensor electrodes100 of each set are sized and shaped to make an electrical connectionwith droplets 1 between which a droplet interface is formed in arespective system of droplets 1. This may be achieved by the area of thesensor electrodes 100 being similar to the area enclosed by the contactline of the droplets 1 with the sensor electrodes 100, and distancebetween the centre of the sensor electrodes 100 within each set andbeing similar to the distance between the centre of the droplets 1 inthe formed system. Each set of sensor electrodes 100 may be aligned witha respective system of droplets 1 for making electrical connections torespective droplets 1 in that system of droplets 1. Thus, the controlsystem 37 may be configured to form plural systems of two or moredroplets 1, where each system of droplets 1 is aligned with a respectiveset of sensor electrodes 100.

By way of illustration, FIG. 7 shows three sets of two sensor electrodes100 and three systems of two droplets 1 formed in alignment with thesensor electrodes 100 of the respective sets. However, in general, therecould be any number of sets of sensor electrodes 100, and the sets couldcontain any number of sensor electrodes 100 in dependence in the numberof droplets 100 to be included in each system.

As a result of this configuration, systems of droplets 1 may be formedin parallel and experiments may be performed thereon in parallel usingthe respective sets of sensor electrodes 100. In general any number ofsystems of droplets 2 may be formed, for example two or more, up tolarge numbers of order tens of thousands.

The conductive tracks 101 are connected to the sensor electrodes 100 andextend to the edge of the layer 58 of conductive material where anelectrical connection is made to a droplet interface sensor system 110described further below. Thus, the conductive tracks 101 provide anelectrical connection from the sensor electrodes 100 to the dropletinterface sensor system 110.

The further electrode 102 extends around the sensor electrodes 100 andthe conductive tracks 101.

The further electrode 102 may function as the reference electrode 59 inthe circuit representations shown in FIGS. 4A and 4B. In that case, thecontrol system 37 is connected to the further electrode 102 and isarranged to apply a reference signal to the further electrode 102, whileapplying actuation signals to the actuation electrodes 48.

However, the further electrode 102 is not essential. When the furtherelectrode 102 is absent, or even when the further electrode 102 ispresent, a different electrode(s) may function as the referenceelectrode 59. In one example, the sensor electrodes 100 may function asthe reference electrode 59.

In another example, a reference electrode 59 may be provided elsewherebetween the first and second substrates 44 and 54, for example as aseparate element such as an in-plane reference electrode. In any suchexample, the control system 37 is connected to the reference electrode59, e.g. the sensor electrodes 100, and is arranged to apply a referencesignal to the reference electrode 59, while applying actuation signalsto the actuation electrodes 48. In such an arrangement, unactuatedactuation electrodes 48 on the first substrate 44 may operate as areference and droplets 1 can be moved without needing a referenceelectrode on the second substrate 54.

The reader 32 further comprises the droplet interface sensor system 110including a measurement unit 111 which is connected to the sensorelectrodes 110 and takes electrical measurements between sensorelectrodes 110 that are electrically connected to respective droplets 1,across droplet interfaces formed therebetween. The measurement unit 111is typically controlled to take electrical measurements out whileactuation signals are not applied to the actuation electrodes 48. Thishas the advantage of reducing the risk of the actuation signalsaffecting the electrical measurements, for example by physicallyaffecting or damaging the system of droplets being measured or bycausing electrical interference with the measurement unit 111.

The elements of the thin film electronics 46 are electrically isolatedfrom the sensor electrodes 100 and the measurement unit 111, so do notparticipate in taking of the electrical measurements.

Any suitable electrical measurements may be taken, for exampleimpedance, current or capacitance measurements. In a possibleconfiguration, the electrical measurements may be taken by applying avoltage and measuring the current sourced through one of the sensorelectrodes 100, whilst the other sensor electrode 100 is grounded. Thereal and imaginary parts of the electrical impedance of the dropletinterface 2 may thus be determined.

The measurement unit 111 may be formed by suitable electronic componentssuitable for droplet interface experiments, for example includingdetection channels including amplifier arrangements. In one example, themeasurement unit 111 may comprise a patch clamp arrangement. In anotherexample, the measurement unit 111 may have the same construction as thesignal processing function described in WO-2011/067559.

The electrical measurements may be taken in a frequency range from alower limit to an upper limit, wherein the lower limit is 1 Hz, 10 Hz or100 Hz and the upper limit is 10 MHz, 100 KHz or 10 KHz, in anycombination.

The measurement unit 111 may be arranged to apply a potential differencebetween a respective pair of sensor electrodes 100 across whichmeasurements are taken, while taking those measurements.

The measurement unit 111 is controlled by the control system 37 to takeelectrical measurements from any of the systems of droplets 1 after theyhave been formed in the AM-EWOD device 34 under the control of thecontrol system 37.

The electrical measurements may be of any suitable type, for examplebeing impedance measurements and/or measurements of ion current flowacross the droplet interface. Where the measurements are taken acrossdroplet interface comprising a membrane of amphipathic molecules havinga transmembrane pore inserted therein, the electrical measurements maybe, for example, measurements of ion current flow between the dropletsthrough the transmembrane pore and/or electrical measurements that aredependent on an analyte that interacts with the transmembrane pore.

The measurements may be optical or a combination of optical andelectrical, such as disclosed by Soni G V et al., Rev Sci Instrum. 2010January; 81(1):014301 and T Gilboa and A Meller, Analyst, 2015, 140,4733-4747.

The droplet interface sensor system 110 may further comprise an analysissystem 112 configured to process the electrical measurements that aredependent on an analyte that interacts with a transmembrane pore, inorder to analyse the analyte. For example, where the analyte is apolymer comprising polymer units, the analysis system may be configuredto process the electrical measurements to derive estimated identities ofthe polymer units of the polymer. The analysis system 112 may processthe electrical measurements using any suitable known technique, someexamples of which are described further below.

The analysis system 112 may be formed by an appropriate combination of(1) a hardware stage, for example a field programmable gate array(FPGA), to pre-process the electrical measurements supplied as a signalfrom the measurement unit 111, and (2) a processor for processing thesignals supplied from the hardware stage. The processor may be anysuitable form of processing device. The processor may be implementedwithin the reader 32 as shown in FIG. 1, and may execute software whichmay be stored in the storage device 40. As an alternative, the processorcould be implemented by a processing device, for example a conventionalcomputer apparatus, external to the reader 32.

By way of example, the measurement unit 111 and the analysis system 112may have the same construction as the signal processing functiondescribed in WO-2011/067559.

The droplet interface sensor system 110 may be combined with other typesof measurement system to take measurements, for example capacitancemeasurements from the actuation electrodes, measurements from additionalelectrodes (not shown) and/or measurements using electro-magneticradiation, including but not limited to absorbance or emission infrared, ultraviolet, which techniques may employ labelled dyes orantibodies, and/or fluorescence resonance energy transfer (FRET).

Droplet Sensing

The array element circuit 72 also may contain a droplet sensor circuit90, which is in electrical communication with the actuation electrode48. The droplet sensor circuit 90 provides a sensing capability fordetecting the presence or absence of a droplet 1 in the location of eachactuation electrode 48. In this manner, the array element circuit 72 mayalso perform a function of sensing the presence or absence of a droplet1 at the location of the array element 51 during manipulation of thedroplets 1. However, due to the presence of the insulating layer 61including layer 62 of electrically insulating material between theactuation electrodes 48 and the droplets 1, it may be difficult orinconvenient to take electrical measurements suitable for studying adroplet interface or processes occurring at a droplet interface.

The droplet sensor circuit 90 may conveniently employ capacitive sensingusing an impedance sensor circuit. The droplet sensor circuit 90 mayinclude impedance sensor circuitry of the type known in the art, asdescribed for example in U.S. Pat. No. 8,653,832 and GB-2,533,952. Asdescribed therein, droplets 1 may be actuated by means ofelectro-wetting and may be sensed by capacitive or impedance sensingmeans. Typically, capacitive and impedance sensing may be analogue andmay be performed simultaneously, or near simultaneously, at every arrayelement 51. By processing the returned information from such a sensor(for example in the application software in the storage device 40 of thereader 32), the control system 37 can determine in real-time, or almostreal-time the position, size, centroid and perimeter of each droplet 1present in the AM-EWOD device.

Alternatively, such sensing may be performed by some other means, forexample optical or thermal means. An alternative to the droplet sensorcircuit 90 is to provide an external sensor such as an optical sensorthat can be used to sense droplet properties, as is known in the fieldof electro-wetting devices.

The control system 37 generates and outputs control signals for thedroplet sensor circuit 90 to perform sensing operations duringmanipulation of the droplets 1. Integrated sensor row addressing 78 andcolumn detection circuits 80 are implemented in the thin filmelectronics 46 for the addressing and readout of the droplet sensorcircuit 90 in each array element circuit 72. Typically, the read-out ofthe droplet sensor circuit 90 may be controlled by one or moreaddressing lines (e.g. RW) that may be common to array elements 51 inthe same row of the array 50, and may also have one or more outputs,e.g. OUT, which may be common to all array elements 50 in the samecolumn of the array 50.

The control system 37 may use the output of the of the droplet sensorcircuit 90 to control the timing of the application of actuation signalsto the actuation electrodes 48 when manipulating droplets 1.

Formation of Droplet Interfaces

The control system 37 is configured to control the AM-EWOD device 34 toform systems of droplets 1 having one or more droplet interfaces 2between pairs of droplets 1 as follows.

Firstly, the control system 37 controls the droplet preparation system35 to form the droplets 1 in the AM-EWOD device 34, as needed forrespective systems of droplets 1. The droplets 1 may be prepared fromany appropriate reagents, as required for the experiments beingperformed. Suitable reagents are described below.

Next, the control system 37 controls the application of actuationsignals to the actuation electrodes 48 to form the systems of droplets1.

It has been considered to simply apply actuation signals to manipulatethe droplets 1 by simply moving the droplets across the array 50 ofactuation electrodes 48 from where they are formed towards each otherand into contact. However, in that case, the droplets 1 have a tendencyto fuse and it is difficult to maintain the droplet interface betweenthe droplets 1. Optimising of conditions to promote formation of adroplet interface 2 is difficult as electro-wetting is dependent onseveral factors which are likely to change between samples, such as saltconcentration, droplet reagents (especially membrane components) anddroplet size.

Accordingly, a different method is implemented employing two stages, aswill now be described.

An example of the method is shown in FIGS. 8 to 12 which shows a planview of a pair of droplets 1 on an array 50 of actuation electrodes 48successively as the droplets are manipulated during the method. Inparticular, FIGS. 8 to 12 show the contact lines of the droplets on thearray 50 of actuation electrodes 48. FIGS. 8 to 12 also show the pattern53 of actuation signals applied, by hashing of the selected actuationelectrodes 48 to which actuation signals are applied 53. This example ismerely for illustration and is not limitative. Various changes may bemade, for example to the size of the droplets 1 and the pattern ofactuation signals may be made. It is also noted that FIGS. 8 to 12relate to an example in which the liquid of the droplets 1 is polar andthe fluid medium 50 is apolar, with the result that the actuationelectrodes 48 to which actuation signals are applied are electro-wet. Ina notional alternative in which the liquid of the droplets 1 is apolarand the fluid medium 50 is polar, then the pattern of actuation signalswould be inverted with the result that the actuation electrodes 48 towhich actuation signals are not applied attract the apolar droplets.

By way of background, it is noted that, in a relaxed state of thedroplets 1 where they are not electro-wet by the application ofactuation signals to the actuation electrodes 48, the droplets 1 wouldtake the shape of lowest surface energy, which would generally be acircular shape where the hydrophobic surface of the insulator layer 61has uniform properties.

In a first stage of the method, actuation signals are applied to theselected actuation electrodes 48 to energise the one, or preferablyboth, of two droplets 1 between which a droplet interface is to beformed. For clarity of description, the case of energising both of thetwo droplets 1 will now be described.

In the energised state, the shape of the droplets 1 is modified comparedto a shape of the droplets 1 in the lower energy state of the droplets1. In such an energised state, the two droplets 1 are moved intoproximity with a gap 3 therebetween. Due to the gap 3, the droplets 1 donot contact each other at this time.

The first stage of the method may be performed under feedback controlfrom the droplet sensor circuit 90.

Examples of the processes applied in the first stage are shown in FIGS.8 to 10, as follows.

FIG. 8 shows a step where a pattern 53 of actuation signals is appliedto a square 4-by-4 group of actuation electrodes 48. This energises thetwo droplets 1 to form a corresponding shape that is generally alsosquare but with some rounding of the corners that minimises the surfaceenergy of droplets 1. FIG. 8 also shows how the droplets 1 may be movedtogether. In particular, FIG. 8 shows the case that the pattern 53 ofactuation signals is applied to a group of actuation electrodes 48 thatis shifted relative to the previous step. This has the result of movingthe droplets 1 towards the group of actuation electrodes 48, in thedirection of the arrows 4. In this manner, the droplets 1 may be shapedand may be moved.

FIG. 9 shows a step where a pattern 53 of actuation signals is appliedto a rectangular 2-by-8 group of actuation electrodes 48. This energisesthe two droplets 1 to form a corresponding shape that is generally alsorectangular but with some rounding of the corners that minimises thesurface energy of the droplets 1. In this step, the rectangular 2-by-8groups of actuation electrodes 48, and hence the droplets 1, are inproximity with a gap 3 of two columns of actuation electrodes 48.

FIG. 10 shows a step subsequent to that shown in FIG. 9 where theactuation signals have the same pattern 53 except that one of the 2-by-8groups of actuation electrodes 48 is shifted by one column of actuationelectrodes 48, so that the rectangular 2-by-8 groups of actuationelectrodes 48, and hence the droplets 1, are in proximity with a gap 3of a single column of actuation electrodes 48. In this example, this isthe final step of the first stage of the method.

In a second stage of the method, the applied actuation signals arechanged such that the energy of the droplets 1 is lowered into a lowerenergy state.

In this stage, the change is preferably to apply no actuation signals toactuation electrodes 48 that affect the droplets 1. In that case, noenergy is supplied to the droplets 1 from the actuation electrodes 48,so the lower energy state is a state of minimum energy of the droplets 1where their shape after relaxation is affected solely by the materialproperties. Alternatively, in the change may in principle be to applyactuation signals that energise the droplets 1 but to a lesser degree,so that the droplets 1 relax and their shape changes but to a lesserdegree than when no actuation signals are applied to actuationelectrodes 48 that affect the droplets 1.

As a result of being placed in a lower energy state, the surfaces of thedroplets 1 that face one another across the gap 3 relax into the gap 3and contact each other, thereby forming a droplet interface 2 betweenthe two droplets 1. Thus, the movement of the surface of the droplets 2is caused by relaxation from the energised state of the droplets 1generated in the first stage. This is a passive process that providesreliable formation of the droplet interface 2. The rate to which adroplet 1 relaxes may be dependent upon one or more factors, such asrelative viscosity of the liquid of the droplet 1 to that of the fluidmedium 50, the size of the droplet 1 and/or the size of the gap 3.

The device geometry (size of droplets 1, height of gap between thehydrophobic surfaces, etc.) and surface tensions at the dropletinterfaces 2, which are themselves dependent on choice of materials andmaterial properties, are arranged such that when the surfaces of thedroplets 1 touch, the droplets 1 do not fuse or coalesce, but rather adroplet interface 2 is formed. The geometry of the patterns of actuationsignals and spatial dimensions of the droplets 1 will typically bearranged such that the droplet interface 2 is formed with a minimalsurface area.

The applied actuation signals may be changed in any manner tode-energise the droplets 1. Where the actuation signals that are appliedto electro-wet the actuation electrodes are AC actuation signals, thenthe change is desirably to replace the AC actuation signals whichenergised the droplets 1 by DC potentials, for example a groundpotential, or by floating potentials. This has the benefit that ACsignals are no longer applied to the actuation signals, which assists informing of the droplet interface 2 because the presence of AC electricfields resulting from AC signals increases the risk of the dropletinterface 2 rupturing and causing the droplets 2 to fuse when thesurfaces of the droplets 2 come into contact.

Other changes which de-energise the droplets 1 may alternatively bemade. An alternative is to remove all power from the array 50 ofactuation electrodes 48. However, it may be preferable to apply a DCpotential to assist in shielding the droplet interface 2 from unwantedenvironmental electro-magnetic interference.

The present inventors have appreciated that it is preferable not tode-actuate the droplets in the conventional way by applying AC voltagewaveforms, since resultant perturbations may damage a droplet interface2 or may interfere with electrical measurements through the dropletinterface 2.

Examples of the processes applied in the second stage are shown in FIGS.11 and 12, as follows.

FIG. 11 shows a step where the pattern of actuation signals is changedcompared to that shown in FIG. 10 by ceasing the application ofactuation signals to the two 2-by-8 groups of actuation electrodes 48and instead applying a DC potential or floating potential. FIG. 11,shows the droplets 1 at the moment where the change is made, when thedroplets instantaneously have the same generally rectangular shape asbefore. However, the droplets then relax into the lower energy stateshown in FIG. 12. In the absence of the other droplet 1, each droplet 1would take its own lower energy state which is generally circular, butthe centre of mass of the droplets 1 remains in generally the samelocation. Thus, in relaxing towards those lower energy states, thesurfaces of the droplets 1 that face one another across the gap 3 relaxinto the gap 3 and contact each other, thereby forming a dropletinterface 2.

The particular shapes of the droplets 2 in the energised state shown inFIGS. 8 to 10 are not limitative, and in general any shape could be usedthat allows relaxation of the droplets 1 into contact to form a dropletinterface. Typically, the shape of the energised contact line of thedroplets 2 may be elongate, with the gap 3 extending along a length ofthe elongate shape. Any elongate shape may be chosen, for example arectangular shape as shown in FIG. 9, an ellipsoidal shape, or a morecomplex shape. Shapes which are not elongate may also be used, forexample a square shape as shown in FIG. 8.

The exact shape of the droplets 1 is selected by control of the patternof the actuation signals, but may vary from that due to the surfacetension between the droplets 1 and the fluid medium 50, which willdepend on the material properties.

Where the shape of the energised contact line of the droplets 2 iselongate, the shape of the energised droplets 1 may have an aspect ratioof at least 2:1, preferably at least 4:1 or at least 8:1. In general,increasing the aspect ratio increases the degree of movement of thesurface of the droplets 1 when they are de-energised, thereby assistingin bringing the droplets 1 into contact.

The gap 3 between the droplets 1 in the first stage is chosen such thatthe two droplets 1 are sufficiently close, although not contacting, thatthey form a droplet interface 2 when placed in the lower energy state.The width of the gap 3 when the droplets 1 are brought into proximity ischosen to allow the droplets 1 to come into contact when the pattern ofactuation signals is changed. This may depend on the same of thedroplets 1 in the energised state. Typically, the width of the gap 3when two droplets 1 are brought into proximity may be chosen so that thecentroids of the two droplets 1 are separated by a distance less thanthe combined radii of the droplets 1 along a line between the twocentroids in the lower energy state of the droplets 1.

The gap 3 may have a width of a single row or column of actuationelectrodes 38 in the array 50, or two or more rows or columns ofactuation electrodes 38 in the array 50.

To assist these processes, the area enclosed by the contact line of thedroplets 1 is desirably large compared to the area of the actuationelectrodes 38. This increases the resolution of the control of the shapein the energised state of the droplets 1, assisting in allowing movementof the droplets 1 across the array and movement of the surfaces of thedroplets 1 into contact on relaxation. The AM-EWOD device 1 is thereforedesigned with actuation electrodes 48 that are sized having regard totypical sizes of droplets 1 desired to be used experimentally. Aparticular advantage of the active matrix arrangement is that it allowsapplication of actuation patters of actuation signals at a resolutionthat is high compared to the size of the droplets 1.

Typically, the area enclosed by the contact line of the droplets 1 inthe lower energy state is at least two times the area of an actuationelectrode 48, preferably at least 5 times, at least 10 times or at least20 times. Thus, the area enclosed by the contact line of the droplets 1in the lower energy state may cover at least two actuation electrodes48, preferably at least 5 actuation electrodes 48, at least 10 actuationelectrodes 48 or at least 20 actuation electrodes 48.

The above description refers to energising both droplets 1 for ease ofdescription, but alternatively a droplet interface 2 may be formed byonly energising one of the two droplets 1 in the first phase. In thatcase, on changing the actuation signals in the second phase, a surfaceof that one droplet 1 relaxes into contact with a stationary surface ofthe other droplet 1.

Systems of Droplets

Above, there is described formation of a single droplet interface 2between a system of two droplets 1. Using similar methods, pluraldroplet interfaces 2 may be formed between respective pairs of dropletsin a system of three or more droplets 1. Droplet interfaces 2 may beformed sequentially by bringing droplets 1 into contact successively orsimultaneously. By way of example, FIG. 13 illustrates an example offorming a system of three droplets 1 having two droplet interfaces 2that are formed sequentially, and FIG. 14 illustrates an example offorming a system of three droplets 1 having two droplet interfaces 2that are formed simultaneously. In each of these examples, the dropletinterfaces 2 are formed using the method described above.

In general, the configuration of the formed system of droplets 1 ischosen in a manner to perform a desired experiment. In such formedsystems, the droplets 1 may be arranged in series with dropletinterfaces 2 similarly in a series, as shown for example in the systemsof three droplets 1 shown in FIGS. 13 and 14. Alternatively, in theformed systems, the droplets 1 may have more complex arrangements orclusters, two non-limitative examples of which are shown in FIGS. 15 and16.

In such systems of droplets 1, the droplets 1 may be or of equal orunequal volume and the droplets 1 may have the same or differentconstituents.

One or more droplets in a system may comprise transmembrane pore capableof insertion into a droplet interface 2. Typically, after formation of adroplet interface 2, the transmembrane pore inserts spontaneously intothe droplet interface 2, after which electrical measurements may betaken. One or more droplets 1 in a system may comprise an analyte thatinteracts with the transmembrane pore.

There are a number of advantages in use of the apparatus 1 to form adroplet interface 2 and subsequently take electrical measurements on thedroplet interface 2 thus formed, in particular when making a system ofthree or more droplets 1 having plural droplet interfaces 2. For examplerelatively small sample volumes may be used as compared to some othertechniques that involve formation of an array of planar membranes. Itallows the possibility of using long lengths of polynucleotide as thereis a reduced chance of shearing as library preparation may occur on thesame device as measurement. Given that a sample does not need to betransferred, the contamination risks are lower. Because all of thesample is contained in either one or both of the droplets 1, there issmall sample loss, which can be recovered. As electro-wetting is usedfor all liquid manipulation, the need for pumps or other moving parts iseliminated. Since droplet positioning is controlled through programmedscripts, sample preparation can be automated. Droplets of DNA sample canthen be supplemented with desired components including polymer vesicles,reagents, pores and analytes.

In one type of experiment, droplets 1 may be periodically split off froma volume of sample, for example to monitor an ongoing reaction occurringin the sample. This provides for analysis with time, titration ofreactant, change in conditions, etc.

Other advantages include:

-   -   the ability to perform coupled library preparation/PCR with        sensing/sequencing; ease of use library to sequence (automated,        walk away)    -   low contamination risks    -   use of compartmentalised samples for library or sequencing    -   permitting sampling different positions of sample/reaction, for        example the length through a gel/mesh/diffusion barrier,        positions on a cell sample and/or a        concentration/thermal/density gradient.

Feedback and Modification

As described above, the apparatus 1 is suitable for forming dropletinterfaces 2 in systems of droplets 1 and performing experiments onthose droplet interfaces 2. Particular advantage is obtained by thecontrol system 37 modifying a formed system of droplets 1 in response tooutputs of the droplet interface sensor system 110. Thus, the system ofdroplets 1 may be modified to modify ongoing performance of theexperiments using feedback from the experiment previously performed.This provides a powerful experimental tool, because the experiments maybe adaptively performed.

Various outputs of the droplet interface sensor system 110 may be usedto provide feedback, for example as follows.

The outputs of the droplet interface sensor system 110 that may be usedinclude electrical measurements taken by the measurement unit 111. Thisprovides a first type of control. As the electrical properties arefundamental to the relevant processes such as formation of dropletinterfaces and reactions occurring there, this first type of controlallows those processes to be considered and adaptively modified. Forexample, electrical measurements taken by the sensor system may be usedto determine whether a droplet interface 2 has been formed successfully.

The outputs of the droplet interface sensor system 110 that may be usedinclude outputs of the analysis system 112. This provides a second typeof control. As such analysis allows higher level information to beobtained, for example concerning an analyte being analysed, this secondtype of control provides powerful experimental adaption based on theresults of the analysis.

The control system 37 may modify a formed system of droplets 1 invarious ways, for example as follows.

The control system 37 may modify a formed system of droplets 1 byseparating a droplet interface 2 between in the system. To do this, thecontrol system 37 applies a pattern of actuation signals to theactuation electrodes 48 that moves apart one or both droplets betweenwhich the droplet interface 2 is formed. The separation of the droplets1 separates the droplet interface 2.

Such separation may be used, for example, to stop an interactionoccurring at the droplet interface 2. This may be done, for example,when the electrical measurements taken by the measurement unit 111indicate that a droplet interface 2 has not been formed successfully orthe outputs of the analysis system 112 indicate that an analysis hasbeen completed, for example because an analyte has become depleted, orsufficient electrical measurements about a particular analyte have beentaken.

The control system 37 may modify a formed system of droplets 1 by movinga new droplet 1 into contact with a current droplet 1 in the system ofdroplets 1 and forming a droplet interface 2 between the new droplet 1and the current droplet 1. To do this, the control system 37 appliesactuation signals to the actuation electrodes 48 using the same methodas described above.

Such formation of a new droplet interface 2 may be used, for example,when the electrical measurements taken by the measurement unit 111indicate that a droplet interface 2 has not been formed successfully soit desired to form a new droplet interface, or the outputs of theanalysis system 112 indicate that an analysis at a droplet interface 2has been completed and it is desired to obtain further measurements.

The control system 37 may modify a formed system of droplets 1 by movinga new droplet 1 into contact with a current droplet 1 in the system ofdroplets 1 and fusing the new droplet 1 and the current droplet 1. To dothis, the control system 37 applies actuation signals to the actuationelectrodes 48 that moves the new droplet 1 into contact with a currentdroplet 1 and causes them to fuse. The fusing of the droplets 1 may beachieved simply by the movement of the new droplet 1 into contact with acurrent droplet 1 without using the method described above to form adroplet interface. Alternatively, or additionally, the fusing of thedroplets 1 may be achieved by applying an AC actuation signal thatruptures the droplet interface that would otherwise be formed betweenthe new droplet 1 and the current droplet.

Such fusing of a new droplet 1 into a current droplet 1 of the systemmay be used, for example, to introduce new reagents into the currentdroplet 1, for example when one member of a redox couple in the one ofthe pairs of droplets 1 has become depleted.

When fusing a new droplet 1 in this manner, it may be that the newdroplet 1 does not comprise amphipathic molecules at the interfacebetween the liquid of the droplet 1 and the fluid medium 50.

These and other ways of modifying the formed system of droplets 1 may beused together in any combination, for example to perform a multi-stageexperiment.

Although some specific applications are described above, these are notlimitative and indeed one of the benefits of the feedback is thatversatility. Some further non-limitative examples of applications are asfollows:

-   -   Automated insertion of transmembrane pores    -   Adaption of a system of droplets 1 to unwanted insertion of        pores or secondary pores    -   Control based on reaction/sample conditions    -   Promotion of droplet interface separation    -   Delivery of more sample or reagent    -   Delivery of a different sample    -   Delivery of more mediator to a droplet    -   Separation of a droplet 1 to take a sample elsewhere and/or to        recover it and/or to return it to an original volume of sample    -   Change of reaction conditions (e.g. temperature, additive,        quench/activate)    -   Taking of an alternative measurement (e.g. absorbance)    -   Return of sample to original volume    -   Performance of a new reaction on an analysed sample (or part        thereof)    -   Control of multiple pore types and balancing of each for        multiple membranes    -   Formation of membrane arrangements with multiple membranes and        pores that interfaces with the same sample    -   Performance of an experiment only until sufficient information        has been obtained, thereby increasing overall experimental        throughput.    -   Queuing/pooling of samples, e.g. allowing delivery of library        samples from a queue of samples on demand, and/or changing the        queue order    -   Pooling of samples as a result of sequencing/sensing    -   Determination of which of plural samples to analyse    -   Determination of duration of run/success criteria    -   Determination of conditions for sample modification prior to        membrane/pore analysis (e.g. type/concentration of library prep)    -   When droplets 1 are periodically split off from a volume of        sample, use of the result of previous experiments as feedback to        adapt reaction/sample conditions    -   Performance of directed evolution using membrane/pore as sensor

Droplets in Fluid Medium

Where reference is herein to droplets comprising liquid in a fluidmedium, the liquid and the fluid medium may be chosen as follows. Ingeneral, any liquid that forms a droplet in a fluid medium may be used,but some possible materials are as follows.

The fluid medium may in principle be a gaseous medium, but is preferablya liquid medium.

In some cases, and often when the fluid medium is a liquid medium, oneof the liquid and the fluid medium is polar, and the other of the liquidand the fluid medium is apolar. Preferably, the liquid of the dropletsis polar, and the fluid medium is apolar.

When one of the liquid and the fluid medium is polar, the polar mediumis typically an aqueous liquid that comprises water. The aqueous liquidmay further comprise one or more solutes. The aqueous liquid may forinstance comprise a buffer in order to regulate the pH of the aqueousmedium as appropriate, and it may comprise a supporting electrolyte. Theaqueous medium may for instance comprise a redox couple, or a member ofa redox couple which may be partially oxidised or reduced to provide theredox couple. The redox couple may chosen from those known in the artsuch as Fe²⁺/Fe³⁺, ferrocene/ferrocenium or Ru²⁺/Ru³⁺. Examples of suchare ferro/ferricyanide, ruthenium hexamine and ferrocene carboxlic acid.

Alternatively, when one of the liquid and the fluid medium is polar, thepolar medium may comprise a polar organic solvent. The polar organicsolvent may for instance be a protic organic solvent, such as analcohol, or it may be an aprotic polar organic solvent.

The liquid of the droplets may be any liquid suitable for performingexperiments of the type described below. Different droplets may comprisedifferent liquids.

Where the other of the liquid and the fluid medium is apolar, then theapolar medium may comprise an oil. The oil may be a single compound, orthe oil may comprise a mixture of two or more compounds.

In one example, the oil is pure alkane hydrocarbon.

The oil may for instance comprise silicone oil. Suitable silicone oilsinclude, for instance, poly(phenyl methyl siloxane) andpoly(dimethylsiloxane) (PDMS). The silicone oil may comprise ahydroxy-terminated silicone oil, for instance hydroxy terminated PDMS.

Additionally or alternatively, the oil may comprise a hydrocarbon, forinstance hexadecane, although any suitable hydrocarbon may be used. Whenthe oil comprises a hydrocarbon it may comprise a single hydrocarboncompound, or a mixture of two or more hydrocarbons. When the oilcomprises a hydrocarbon, the hydrocarbon may be branched or unbranched.The hydrocarbon may for instance be squalene, hexadecane or decane. Inone embodiment it is hexadecane. However, in some embodiments thehydrocarbon may be substituted with a halogen atom, for instancebromine.

The oil may comprise a mixture of one or more silicone oils and one ormore hydrocarbons. The silicone oil and hydrocarbon in the mixture mayboth be as further defined above. The silicone oil may for instance bepoly(phenyl methyl siloxane) or PDMS.

Other types of oil are also possible. For example, the oil may be afluorocarbon or a bromo-substituted C₁₀-C₃₀ alkane.

Amphipathic Molecules

In the case that one of the liquid and the fluid medium is polar, andthe other of the liquid and the fluid medium being apolar, then thedroplets may further comprise amphipathic molecules at the interfacebetween the liquid of the droplets and the fluid medium. Suchamphipathic molecules serve to stabilise the droplets in the fluidmedium prior to formation of a droplet interface. Also, the amphipathicmolecules may allow the droplet interface, when formed, to comprise amembrane of amphipathic molecules.

Numerous different types of amphipathic molecules may be used. Somenon-limitative examples of types of amphipathic molecules that may beused are as follows.

In one example, the amphipathic molecules may comprise a lipid, whichmay have a single component or a mixture of components, as isconventional when forming lipid bilayers.

Any lipids that form a membrane such as a lipid bilayer may be used. Thelipids are chosen such that a lipid bilayer having the requiredproperties, such as surface charge, ability to support membraneproteins, packing density or mechanical properties, is formed. Thelipids can comprise one or more different lipids. For instance, thelipids can contain up to 100 lipids. The lipids preferably contain 1 to10 lipids. The lipids may comprise naturally-occurring lipids and/orartificial lipids.

The lipids can also be chemically-modified.

Amphipathic polymer membranes are preferred over lipid membranes due totheir ability to withstand higher voltages.

In another example, the amphipathic molecules may comprise anamphipathic compound comprising a first outer hydrophilic group, ahydrophobic core group, and a second outer hydrophilic group, whereineach of the first and second outer hydrophilic groups is linked to thehydrophobic core group.

Some such amphipathic compounds are disclosed in WO 2014/064444.

Other such amphipathic compounds are disclosed in U.S. Pat. No.6,916,488 which is incorporated herein by reference and discloses anumber of polymeric materials that can be employed in the apparatus 1 asplanar amphipathic membranes. In particular triblock copolymers aredisclosed, for example silicon triblock copolymer membranes such aspoly(2-methyloxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyloxazoline)(PMOXA-PDMS-PMOXA).

Examples of silicone triblock polymers that may be employed are 7-22-7PMOXA-PDMS-PMOXA, 6-45-6 PMOXA-PE-PMOXA and 6-30-6 PMOXA-PDMS-PMOXA,where the nomenclature refers to the number of subunits.

Such triblock copolymers may be provided in vesicle form in thedroplets.

Depending on the nature of the amphipathic molecules, the membranes maybe bilayers of the amphipathic molecules or may be monolayers of theamphipathic molecules.

The amphipathic molecules may alternatively be replaced by anothersurfactant.

Different droplet interfaces may comprise membranes of differentamphipathic molecules, for example membranes comprising a lipid bilayerand a polymer membrane such as a silicone triblock polymer membrane asdescribed above, such as disclosed in WO2017/004504.

The electrical measurements that are taken may be used to study themembrane of amphipathic molecules itself, or interactions thereof, forexample to study drug-membrane permittivity.

Transmembrane Pore

In general any transmembrane pore may be used that is capable ofinserting into the droplet interface. Different droplets may comprisethe same or different transmembrane pore, so that when plural dropletinterfaces are formed between different plural droplet pairs, the sameor different transmembrane pore may insert into those dropletinterfaces.

Some non-limitative examples of types of transmembrane pore that may beused are as follows.

A transmembrane pore is a channel structure that provides a pathway fromone of a membrane to the other through which ions may flow. The channelmay vary in width along its length and typically has an inner diameterof between 0.5 nm and 10 nm.

Any suitable transmembrane pore may be used in the invention. The poremay be biological or artificial. Suitable pores include, but are notlimited to, protein pores, polynucleotide pores and solid state pores.The pore may be a DNA origami pore (Langecker et al., Science, 2012;338: 932-936). Suitable DNA origami pores are disclosed inWO2013/083983.

The transmembrane pore is preferably a transmembrane protein pore.

The transmembrane protein pore may be a monomer or an oligomer. The poremay be a hexameric, heptameric, octameric or nonameric pore. The poremay be a homo-oligomer or a hetero-oligomer.

The transmembrane protein pore i may be derived from CsgG, such as fromCsgG from E. coli Str. K-12 substr. MC4100. Examples of suitable CsgGpores are described in WO-2016/034591, WO-2017/149316, WO-2017/149317and WO-2017/149318.

The transmembrane protein pore typically comprises a barrel or channelthrough which the ions may flow. The subunits of the pore typicallysurround a central axis and contribute strands to a transmembrane βbarrel or channel or a transmembrane α-helix bundle or channel.

The barrel or channel of the transmembrane protein pore comprises aminoacids that facilitate interaction with an analyte, such as a nucleotide,polynucleotide or nucleic acid. The pore may be modified by for examplesubstitution or deletion of one of more amino acids.

Transmembrane protein pores for use in accordance with the invention canbe derived from β-barrel pores or α-helix bundle pores. β-barrel porescomprise a barrel or channel that is formed from β-strands. Suitableβ-barrel pores include, but are not limited to, β-toxins, such asα-hemolysin, anthrax toxin and leukocidins, and outer membraneproteins/porins of bacteria, such as Mycobacterium smegmatis porin(Msp), for example MspA, MspB, MspC or MspD, CsgG, outer membrane porinF (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase Aand Neisseria autotransporter lipoprotein (NalP) and other pores, suchas lysenin. α-helix bundle pores comprise a barrel or channel that isformed from α-helices. Suitable α-helix bundle pores include, but arenot limited to, inner membrane proteins and a outer membrane proteins,such as WZA and ClyA toxin.

The transmembrane pore may be derived from or based on Msp, α-hemolysin(α-HL), lysenin, CsgG, ClyA, Sp1 and haemolytic protein fragaceatoxin C(FraC). The transmembrane protein pore is preferably derived from CsgG,more preferably from CsgG from E. coli Str. K-12 substr. MC4100.

The transmembrane pore may be derived from lysenin. Suitable poresderived from lysenin are disclosed in WO 2013/153359.

The pore may be a variant of the above listed nanopores. The variant maybe at least 55%, at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90% and more preferably at least95%, 97% or 99% homologous based on amino acid similarity or identity tothe amino acid sequence.

Standard methods in the art may be used to determine homology. Forexample the UWGCG Package provides the BESTFIT program which can be usedto calculate homology, for example used on its default settings(Devereux et al (1984) Nucleic Acids Research 12, p 387-395). The PILEUPand BLAST algorithms can be used to calculate homology or line upsequences (such as identifying equivalent residues or correspondingsequences (typically on their default settings)), for example asdescribed in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S. Fet al (1990) J Mol Biol 215:403-10. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/). Similarity canbe measured using pairwise identity or by applying a scoring matrix suchas BLOSUM62 and converting to an equivalent identity. Since theyrepresent functional rather than evolved changes, deliberately mutatedpositions would be masked when determining homology. Similarity may bedetermined more sensitively by the application of position-specificscoring matrices using, for example, PSIBLAST on a comprehensivedatabase of protein sequences. A different scoring matrix could be usedthat reflect amino acid chemico-physical properties rather thanfrequency of substitution over evolutionary time scales (e.g. charge).

Amino acid substitutions may be made to the amino acid sequence of SEQID NO: 3, for example up to 1, 2, 3, 4, 5, 10, 20 or 30 substitutions.Conservative substitutions replace amino acids with other amino acids ofsimilar chemical structure, similar chemical properties or similarside-chain volume. The amino acids introduced may have similar polarity,hydrophilicity, hydrophobicity, basicity, acidity, neutrality or chargeto the amino acids they replace. Alternatively, the conservativesubstitution may introduce another amino acid that is aromatic oraliphatic in the place of a pre-existing aromatic or aliphatic aminoacid.

Any of the proteins described herein, such as the transmembrane proteinpores, may be made synthetically or by recombinant means. For example,the pore may be synthesised by in vitro translation and transcription(IVTT). The amino acid sequence of the pore may be modified to includenon-naturally occurring amino acids or to increase the stability of theprotein. When a protein is produced by synthetic means, such amino acidsmay be introduced during production. The pore may also be alteredfollowing either synthetic or recombinant production.

Any of the proteins described herein, such as the transmembrane proteinpores, can be produced using standard methods known in the art.Polynucleotide sequences encoding a pore or construct may be derived andreplicated using standard methods in the art. Polynucleotide sequencesencoding a pore or construct may be expressed in a bacterial host cellusing standard techniques in the art. The pore may be produced in a cellby in situ expression of the polypeptide from a recombinant expressionvector. The expression vector optionally carries an inducible promoterto control the expression of the polypeptide. These methods aredescribed in Sambrook, J. and Russell, D. (2001). Molecular Cloning: ALaboratory Manual, 3rd Edition. Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.

The pore may be produced in large scale following purification by anyprotein liquid chromatography system from protein producing organisms orafter recombinant expression. Typical protein liquid chromatographysystems include FPLC, AKTA systems, the Bio-Cad system, the Bio-RadBioLogic system and the Gilson HPLC system.

Analyte

The droplets may comprise an analyte that is capable of interaction withthe transmembrane pore, also referred to as a target analyte, thetemplate analyte or the analyte of interest. For example, the analytemay be a polymer or a drug.

Electrical measurements that are taken may be dependent on theinteraction of the analyte with the transmembrane pore. The electricalmeasurements may be measurements of ion current through the pore.

Where the electrical measurements are dependent on the interaction ofthe analyte with the transmembrane pore, the analysis may determine thepresence, absence or one or more characteristics of a target analyte.The analysis may determine the presence, absence or one or morecharacteristics of a target analyte. Where the analyte is a polymercomprising polymer units, in the analysis the electrical measurementsmay be processed to derive estimated identities of the polymer units, orto count polymer units or determine length of the polymer. Controlexperiments can be carried out in the presence of different analytes orpolymer units, to determine how analytes affect the electricalmeasurements as the basis for the analysis.

The analysis may be performed using any suitable known technique,including techniques employing a Hidden Markov Model, for example asdescribed in WO-2013/041878 or WO-2015/140535; techniques employingmachine learning for example as described in Boza et al., “DeepNano:Deep recurrent neural networks for base calling in MinION nanoporereads”, PLoS ONE 12(6): e0178751, 5 Jun. 2017; techniques employingcomparison of feature vectors for example as described inWO-2013/121224; or any other suitable technique.

Such interaction may occur as an analyte moves with respect to, such astranslocating through, the pore. In that case, the electricalmeasurements may be taken as the analyte moves with respect to the pore.Such movement may occur while a potential difference is applied betweenthe droplets, i.e. across the pore. The applied potential typicallyresults in the formation of a complex between the pore and apolynucleotide binding protein. The applied potential difference may bea voltage potential. Alternatively, the applied potential difference maybe a chemical potential. An example of this is using a salt gradientacross an amphiphilic layer. A salt gradient is disclosed in Holden etal., J Am Chem Soc. 2007 Jul. 11; 129(27):8650-5.

The target analyte may be a metal ion, an inorganic salt, a polymer, anamino acid, a peptide, a polypeptide, a protein, a nucleotide, anoligonucleotide, a polynucleotide, a dye, a bleach, a pharmaceutical, adiagnostic agent, a recreational drug, an explosive or an environmentalpollutant.

The analyte may be an amino acid, a peptide, a polypeptides and/or aprotein. The amino acid, peptide, polypeptide or protein can benaturally-occurring or non-naturally-occurring. The polypeptide orprotein can include within them synthetic or modified amino acids. Anumber of different types of modification to amino acids are known inthe art. Suitable amino acids and modifications thereof are above. Forthe purposes of the invention, it is to be understood that the targetanalyte can be modified by any method available in the art.

The analyte protein can be an enzyme, an antibody, a hormone, a growthfactor or a growth regulatory protein, such as a cytokine. The cytokinemay be selected from interleukins, preferably IFN-1, IL-1, IL-2, IL-4,IL-5, IL-6, IL-10, IL-12 and IL-13, interferons, preferably IL-γ, andother cytokines such as TNF-α. The protein may be a bacterial protein, afungal protein, a virus protein or a parasite-derived protein.

The target analyte may be a nucleotide, an oligonucleotide or apolynucleotide. Nucleotides and polynucleotides are discussed below.Oligonucleotides are short nucleotide polymers which typically have 50or fewer nucleotides, such 40 or fewer, 30 or fewer, 20 or fewer, 10 orfewer or 5 or fewer nucleotides. The oligonucleotides may comprise anyof the nucleotides discussed below, including the abasic, and modified,nucleotides.

At least a portion of the polynucleotide may be double stranded.

The polynucleotide can be a nucleic acid, such as deoxyribonucleic acid(DNA) or ribonucleic acid (RNA). The polynucleotide can comprise onestrand of RNA hybridised to one strand of DNA.

The polynucleotide can be any length. For example, the polynucleotidecan be at least 10, at least 50, at least 100, at least 500 nucleotidesor nucleotide pairs in length. The polynucleotide can be 1000 or more,10000 or more, 100000 or more, or 1000000 or more nucleotides ornucleotide pairs in length.

Any number of polynucleotides can be investigated. For instance, themethod of the invention may concern characterising 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 50, 100 or more polynucleotides. If two or morepolynucleotides are characterised, they may be different polynucleotidesor two instances of the same polynucleotide.

The polynucleotide can be naturally occurring or artificial.

Where the analyte is a polynucleotide comprising nucleotides andestimated identities of the polymer units are derived from theelectrical measurements, then strand characterisation/sequencing orexonuclease characterisation/sequencing may be applied.

In strand sequencing, the polynucleotide may be translocated through thenanopore either with or against an applied potential. In this case, theelectrical measurements are indicative of one or more characteristics ofmultiple nucleotides.

The droplets may contain a polymer binding moiety such as an enzyme tocontrol translocation of the polymer through the pore. The moiety can bea molecular motor using for example, in the case where the moiety is anenzyme, enzymatic activity, or as a molecular brake.

Where the polymer is a polynucleotide there are a number of methodsproposed for controlling the rate of translocation including use ofpolynucleotide binding enzymes. Suitable enzymes for controlling therate of translocation of polynucleotides include, but are not limitedto, polymerases, helicases, exonucleases, single stranded and doublestranded binding proteins, and topoisomerases, such as gyrases. Forother polymer types, moieties that interact with that polymer type canbe used. The polymer interacting moiety may be any disclosed inWO-2010/086603, WO-2012/107778, and Lieberman K R et al, J Am Chem Soc.2010; 132(50):17961-72), and for voltage gated schemes (Luan B et al.,Phys Rev Lett. 2010; 104(23):238103).

The polymer binding moiety can be used in a number of ways to controlthe polymer motion. The moiety can move the polymer through the nanoporewith or against the applied field. The moiety can be used as a molecularmotor using for example, in the case where the moiety is an enzyme,enzymatic activity, or as a molecular brake. The translocation of thepolymer may be controlled by a molecular ratchet that controls themovement of the polymer through the pore. The molecular ratchet may be apolymer binding protein. For polynucleotides, the polynucleotide bindingprotein is preferably a polynucleotide handling enzyme.

Preferred polynucleotide handling enzymes are polymerases, exonucleases,helicases and topoisomerases, such as gyrases. A polynucleotide handlingenzyme may be for example one of the types of polynucleotide handlingenzyme described in WO-2015/140535 or WO-2010/086603.

In an embodiment, one or more of the membranes may be a selectivemembrane having multiple pores inserted to provide a kind of fritalternative to supply a reagent. This embodiment may be employed forexample in a three pore system comprising a droplet pair and a thirddroplet connected to a droplet of the pair, whereby the interfacebetween the third droplet and the droplet of the pair comprises multiplepores. The third droplet may for example comprise an electrochemicalmediator such as ferricyanide [Fe(CN)₆]^(3−/2−).

Different droplet interfaces may have different transmembrane poresinserted thereon.

Coupling

The analyte may contain an anchor to couple it to a membrane, or atether to couple it to a pore. The membrane may be functionalised tofacilitate coupling of an analyte. The pore may be modified tofacilitate tethering of the analyte. Methods of coupling an analyte to amembrane that are known in the art may be used, for example as describedin WO-2012/164270 or WO-2015/150786. Methods of tethering an analyte toa pore that are known in the art may be used, for example as describedin WO-2012/164270 or PCT/GB2017/053603.

Sample

Droplets 1 may be prepared from a sample. Such a sample may be known tocontain or suspected to contain an analyte

The sample may be a biological sample. The sample may be obtained fromor extracted from any organism or microorganism.

The sample may be obtained from or extracted from any virus.

The sample is preferably a fluid sample. The sample typically comprisesa body fluid of the patient. The sample may be urine, lymph, saliva,mucus or amniotic fluid but is preferably blood, plasma or serum.

The sample may be human in origin, but alternatively it may be fromanother mammal animal such as from commercially farmed animals such ashorses, cattle, sheep, fish, chickens or pigs or may alternatively bepets such as cats or dogs. Alternatively, the sample may be of plantorigin, such as a sample obtained from a commercial crop, such as acereal, legume, fruit or vegetable.

The sample may be or derived from a non-biological sample. Thenon-biological sample is preferably a fluid sample. Examples ofnon-biological samples include surgical fluids, water such as drinkingwater, sea water or river water, and reagents for laboratory tests.

Example

An Example of use of the apparatus 1 which has been carried out is asfollows.

The apparatus 1 of the Example was designed to perform DNA samplepreparation and sequencing in one, portable platform. The key sequencingelement is a protein nanopore embedded in a polymer membrane that isformed at the droplet interface 2 between two aqueous droplets 1 in anAM-EWOD device 34 of the type described above.

In this Example, the liquid of the droplets 1 was aqueous solution, thedroplets contained amphipathic molecules that were triblock copolymersof the type describe above in vesicle form, and the fluid medium 50 waspure alkane hydrocarbon.

The Example used an AM-EWOD device 31 having an array 50 of actuationelectrodes 48 as shown in FIG. 17 which are images also showing twodroplets 1. FIG. 17a was taken at the end of the first stage of themethod described above with the droplets 1 in the energised state inproximity with the gap 3 therebetween. FIG. 17b was taken after the endof the second stage when the droplets 1 have relaxed to form a dropletinterface 2. Each actuation electrode 48 in the array was of dimensions200×200 μm and thus much smaller than any droplet 1 used.

In the AM-EWOD device 31, the layer 58 of conductive material ispatterned as shown in FIG. 18, wherein the box 120 shows the regionwhere the images of FIG. 17 were taken. FIG. 18 shows contact pads 121labelled C1 through C15 at the top edge. A sensor electrode 100connected to the contact pads 121 labelled C2 (grounded) and a sensorelectrode 100 connected to the contact pads 121 labelled C5 (recording)were used for electrical recording from droplet interface 2 in panelFIG. 17 b.

Recording electrodes were integrated into the AM-EWOD device 31 tofacilitate voltage application and the current recording that comprisesthe pore DNA sequencing signal.

In the Example, current recording was performed on individual dropletinterfaces 2 using a standard patch clamp amplifier. For multichannelrecording from an array of droplet interfaces 2 in parallel, amultichannel recording system can be employed. To enable recording ofsufficient quality for DNA sequencing (<1 pA rms @ 5 KHz), the systemmust be virtually free of electrical noise. Therefore, preferentially,the apparatus 1 operates in two mutually exclusive modes, referred to asan EWOD mode and a recording mode.

In the EWOD mode, all features in the layer 58 of conductive materialare connected to the control electronics 38 which supplies a part of thevoltage necessary for movement. Because EWOD uses high frequency, largeAC voltage fields, recording cannot take place while the EWOD field ison. Specifically, the EWOD field generates noise that obscures the DNAsignal. Therefore, once the droplets 1 are positioned as desired, thecontrol electronics 38 is unplugged, although internal switchingcomponents could alternatively be used. Once EWOD is unplugged,multipole switches are actuated

The entire apparatus 1 was enclosed in a Faraday cage during recordingto prevent interference from ambient noise. Thus, during recording mode,the droplets are not held in place by any electrically induced forces.

Using the method described above, the AM-EWOD device 34 was used tocreate three systems each consisting of two droplets 1 having a dropletinterfaces 2 therebetween, as shown in FIG. 19.

Formation of droplet interfaces 2 was performed as follows.

Simply bringing manipulating two droplets 1 to bring them together underthe application of actuation signals to the actuation electrodes 48 waspossible, but challenging because the droplets 1 tended to fuse.

Instead, the method described above was used. Specifically, in the firststage, droplets 1 were energised into rectangular shapes with an aspectratio greater than 1.5. The long edges of these shapes were brought into1-2 pixel proximity and centered. An example of this stage applied tothree droplets 1 is shown in FIG. 20, left hand side.

In the second stage, the actuation signals were switched. The dropletsnaturally relaxed back into relaxed circular shapes to reduce theirsurface area to volume ratio. Relaxation causes the surfaces of thedroplets facing each other across the gaps 3 to contact and form adroplet interface 2 (which may be referred to as passive formation).Using this approach, DIBs may be created between two or more droplets.An example of this stage applied to three droplets 1 is shown in FIG.20, right hand side.

Such formation of droplet interfaces 2 was a reversible process.

Although the control electronics 38 produced noise that would obscurethe DNA sequencing signal, it is still possible to observe a largecurrent event, such as pore insertion into the droplet interface 2. As ademonstration, the apparatus 1 was set up such that the AM-EWOD device34 could be powered to a low noise mode and droplets were positioned toform a droplet interface while recording electrical current.

FIG. 21 shows the electrical current thus recorded. Within this signalit is possible to observe formation of droplet interfaces 2, unzipping,reformation and pore insertion. Multiple cycles of pore insertion andmembrane disconnect and reconnect are shown. Thick black arrows denotepore insertion. The recording time bars indicate recording mode, theEWODon time bars represent EWOD mode and the actuation time barsindicate droplet actuation and shaping. Off-scale noise is observedduring droplet actuation.

Pore insertion was observed as a jump in current from 0 to ˜200 pA at300 mV. After pore insertion, voltage was switched to zero and thesystem switched to EWOD mode. Droplets 1 were separated, and then adroplet interface was reformed. Note that during this time, the noisegoes beyond the scale of the current recording instrument.

The system was then switched back to recording mode and a voltage of 300mV applied. After observing another pore insertion, the cycle wasrepeated once more for a total of three pore insertions and twoseparations of droplet interfaces 2. This demonstrates the ability toform droplet interfaces 2, insert pores, separate droplets 1 and reformdroplet interfaces 2 repeatedly in the AM-EWOD device 34.

DNA detection and sequencing was performed as follows.

By placing the amphipathic molecules in the droplets 1 rather than thefluid medium 50, it becomes possible to make asymmetric membranes. Forexample, the DNA droplet could have a lower concentration of polymervesicle relative to the opposing droplet or it could have an entirelydifferent polymer composition. This may provide flexibility inoptimizing sample prep, DIB formation, pore insertion, DNA sequencing orfurther processes.

In one example, an asymmetric pair of droplets 1 was used as shown inFIG. 22 to detect short DNA strands (adapter). FIG. 22 shows an exampleof droplet interface 2 comprising a membrane of amphipathic moleculeshaving a concentration below 2 mg/mL in the DNA droplet (top) to aidspore insertion. Higher polymer concentration in the opposing droplet(bottom) aids stability.

FIG. 23 shows an example of the characteristic adapter signal obtainedfrom the droplet interface in FIG. 22. Current levels, shown in pA,represent the open pore (2) and an adapter-occupied pore. In FIG. 23,the characteristic squiggle of the adapter blockade is an easilyrecognizable signal that established the quality of the pore and overallsystem configuration.

The same approach can be applied to obtain sequencing signals fromsingle strands of DNA. A droplet interface 2 was created from a droplet1 containing a 3.6 Kb single-strand DNA sample, sequencing reagents andenzymes, polymer vesicles, mediator buffer and nanopores. The opposingdroplet 1 contained vesicles of amphipathic molecules and mediator+saltsto osmotically balance with the DNA droplet 1.

After observing a single pore insertion, the control electronics 38 wereunplugged and electrodes switched to recording mode. During sequencing,a strand of DNA threads into the pore which is then pulled through bythe applied voltage. The speed of threading is regulated by an attachedenzyme that is, in turn, powered by ATP turnover in the droplet.

The DNA used in this experiment was a standard 3.6 Kb long with a knownsequence, so each stand was expected to thread through the pore for asimilar amount of time. FIG. 24 shows an example of the electricalmeasurements taken, showing DNA threading events at 180 mV. Note thatthe current blockades last from 15.1 to 18.3 seconds, which correlatesto roughly 200 bases per second. This is the translocation speedexpected for a nanopore operating under the conditions of theexperiment. Since each DNA strand in the control sample is identical,the squiggle sequence from each translocation event should be the same.

FIG. 25 is a plot of expanded current traces for six translocationevents, each showing a characteristic “a-basic” peak followed by thesequencing signal. Note that all traces possess the same profile. Arough alignment of these current traces of six translocation eventsshows that the signal pattern is the same for each strand of DNA.

1. A method of forming a droplet interface in an electro-wetting device,the electro-wetting device comprising: an array of actuation electrodes;an insulator layer covering the actuation electrodes and having anoutermost hydrophobic surface; disposed on the hydrophobic surface, afluid medium and two droplets comprising liquid in the fluid medium, oneof the liquid and the fluid medium being polar, and the other of theliquid and the fluid medium being apolar, whereby the actuationelectrodes are capable of electro-wetting the droplets when actuationsignals are applied thereto, the method comprising: applying actuationsignals to selected actuation electrodes to place one or both of the twodroplets in an energised state in which the shape of said one or bothdroplets is modified compared to when in a lower energy state and tobring the two droplets into proximity with a gap therebetween, the gapbeing chosen such that the two droplets do not contact each other whenone or both are in the energised state and contact each other to form adroplet interface when in the lower energy state; and changing theactuation signals applied to the actuation electrodes to lower theenergy of said one or both droplets into the lower energy state so thatsaid one or both droplets relax into the gap and the two dropletscontact each other thereby forming a droplet interface.
 2. A methodaccording to claim 1, wherein both droplets are placed in the energisedstate.
 3. A method according to claim 1, wherein the shape of thedroplet is elongate, said gap extending along a major length of theelongate shape.
 4. A method according to claim 3, wherein the shape ofthe droplet has an aspect ratio of at least 2:1.
 5. A method accordingto claim 1, wherein, during the step of applying actuation signals tothe actuation electrodes, the two droplets are brought into proximitywith the centroids of the two droplets separated by a distance less thanthe combined radii of the droplets along a line between the twocentroids in the lower energy state of the droplets.
 6. A methodaccording to claim 1, wherein the area enclosed by the contact line ofthe droplets in the lower-energy state covers at least two actuationelectrodes, preferably at least 5 actuation electrodes, at least 10actuation electrodes or at least 20 actuation electrodes.
 7. A methodaccording to claim 6, further comprising taking electrical measurementsbetween the droplets across the droplet interface, and wherein the stepof taking electrical measurements is carried out while not applyingactuation signals to the actuation electrodes.
 8. (canceled)
 9. A methodaccording to claim 7, wherein the electrical measurements aremeasurements of ion flow between droplets through a transmembrane pore.10. A method according to claim 9, wherein the transmembrane pore is aprotein.
 11. A method according to claim 7, wherein the electricalmeasurements are taken while applying a potential difference between thedroplets.
 12. A method according to claim 11, wherein the actuationsignals applied to the selected actuation electrodes are AC actuationsignals.
 13. A method according to claim 12, wherein the step ofchanging the actuation signals applied to the selected actuationelectrodes comprises applying DC potentials or floating potentials tothe selected actuation electrodes in place of the AC actuation signals.14-15. (canceled)
 16. A method according to claim 1, wherein thedroplets further comprise amphipathic molecules at the interface betweenthe liquid of the droplets and the fluid medium, and the dropletinterface comprises a membrane of amphipathic molecules.
 17. A methodaccording to claim 16, wherein at least one of the droplets comprises atransmembrane pore and the method further comprises allowing thetransmembrane pore to insert into the membrane.
 18. A method accordingto claim 1, wherein the two droplets have respective volumes between 1nL and 10 μL.
 19. A method according to claim 1, wherein the insulatorlayer comprises a layer of electrically insulating material coated by ahydrophobic material that forms said hydrophobic surface.
 20. A methodaccording to claim 1, further comprising a second substrate facing thehydrophobic surface of the insulator layer, wherein the second substrateis coated by a hydrophobic material forming a further hydrophobicsurface facing the hydrophobic surface of the insulator layer, thedroplets being disposed on the further hydrophobic surface of thehydrophobic layer as well as the hydrophobic surface of the insulatorlayer.
 21. A method according to claim 20, wherein the second substratesupports sensor electrodes that make an electrical connection with thedroplets between which a droplet interface is formed.
 22. A methodaccording to claim 21, further comprising taking electrical measurementsbetween the droplets across the droplet interface and wherein theelectrical measurements are made using the sensor electrodes.
 23. Amethod according to claim 22, wherein the electrical measurements aremade using the sensor electrodes while applying a potential differencebetween the sensor electrodes.
 24. A method according to claim 21,further comprising, while applying actuation signals to the actuationelectrodes, applying a reference signal to the sensor electrodes.
 25. Amethod according to claim 20, wherein the second substrate furthersupports at least one further electrode, and the method furthercomprises, while applying actuation signals to the actuation electrodes,applying a reference signal to the further electrode.
 26. A methodaccording to claim 1, wherein the electro-wetting device furthercomprises a further electrode, and the method further comprises, whileapplying actuation signals to the actuation electrodes, applying areference signal to the further electrode.
 27. A method according toclaim 1, wherein the electro-wetting device further comprises an activematrix arrangement connected to the actuation electrodes.
 28. A methodaccording to claim 1, wherein one or more further droplets are disposedon the hydrophobic surface, and the steps of applying actuation signalsto selected actuation electrodes and changing the actuation signalsapplied to the selected actuation electrodes are performed to formplural droplet interfaces between plural pairs of droplets.
 29. A methodaccording to claim 1, wherein the electro-wetting device comprises adroplet preparation system configured to form droplets disposed on thehydrophobic surface, the method further comprising forming the dropletsdisposed on the hydrophobic surface using the droplet preparationsystem.
 30. An electro-wetting device for forming a droplet interface,the electro-wetting device comprising: an array of actuation electrodes;an insulator layer covering the actuation electrodes and having anoutermost hydrophobic surface on which may be disposed a fluid mediumand two droplets comprising liquid in the fluid medium, one of theliquid and the fluid medium being polar, and the other of the liquid andthe fluid medium being apolar, the droplets further comprisingamphipathic molecules at the interface between the liquid of thedroplets and the fluid medium, whereby the actuation electrodes arecapable of electrowetting such droplets when actuation signals areapplied thereto; and a control system the control system beingconfigured to apply actuation signals to selected actuation electrodesto place one or both of the two droplets in an energised state in whichthe shape of said one or both droplet is modified compared to when in alower energy state and to bring the two droplets into proximity with agap therebetween, the gap being chosen such that the two droplets do notcontact each other when one or both are in the energised state andcontact each other to form a droplet interface when in the lower energystate, and the control system being configured to change the actuationsignals applied to the selected actuation electrodes to lower the energyof said one or both droplets into the lower energy state so that saidone or both droplets relax into the gap and the two droplets contacteach other thereby forming a droplet interface between the two droplets.31.-94. (canceled)